The delivery of hydrophilic macromolecules runs into difficulties such as penetration of the cell membrane lipid bilayer. Our prior experiment demonstrated that capsaicin induces the reversible opening of tight junctions (TJs) and enhances the delivery of hydrophilic macromolecules through a paracellular route. Herein, we screened paracellular permeability enhancers other than capsaicin. As TJ opening by capsaicin is associated with Ca2+ influx, we first screened the compounds that induce Ca2+ influx in layered MDCK II cells, and then we determined the compounds’ abilities to open TJs. Our results identified several natural compounds with α,β-unsaturated moiety. A structure-activity relationship (SAR) analysis and the results of pretreatment with reducing reagent DTT suggested the importance of α,β-unsaturated moiety. We also examined the underlying mechanisms, and our findings suggest that the actin reorganization seen in capsaicin treatment is important for the reversibility of TJ opening. Furthermore, our analyses revealed that TRPA1 is involved in the Ca2+ influx and TJ permeability increase not only by an α,β-unsaturated compound but also by capsaicin. Our results indicate that the α,β-unsaturated moiety can be a potent pharmacophore for TJ opening.

MA026 is an antiviral natural compound against hepatitis C virus (HCV). It was recently reported that MA026 binds claudin-1 (CLDN1) and inhibits HCV infection. Although CLDN1 is an important component of tight junctions (TJ) in the epithelial cell layer, the effects of MA026 on the TJ barrier function remained to be revealed. Here we report that MA026 irreversibly opens the TJ. MA026 irreversibly increased FD4 permeability and decreased transepithelial electrical resistance (TER) for at least 5 h. Although MA026 increased Ca 2+ influx in layered MDCKII cells, the Ca 2+ influx was less than that of capsaicin, a reversible TJ opener. Moreover, MA026 did not induce the dephosphorylation of cofilin and reorganization of F-actin structure. Although the mechanism is left to be disclosed, these results suggest that MA026 is a novel irreversible TJ opener probably by targeting CLDN1.